A role for ABCB1 in prognosis, invasion and drug resistance in ependymoma

Abstract

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05–0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.

BLBP Is Both a Marker for Poor Prognosis and a Potential Therapeutic Target in Paediatric Ependymoma

“…In these experiments, we employed the PPAR-γ antagonist GW9662, which had a consistent inhibitory effect on BLBP expression and survival in both cell lines. In a stem cell-relevant clonogenic assay, pre-treatment of the BLBPlo BXD-1425EPN cell line with GW9662 for 48 h followed by treatment with etoposide and vincristine at their IC50 values [ 5 ] potentiated response to both drugs ( Figure 5 a(i,ii)).…”

“…The CNS9904 cohort consisted of patients who were only given chemotherapy if incomplete resection was determined after a second attempt at surgery. Clinicopathological details of both these trials have been previously described [ 5 , 36 ]. Sections from the mouse orthotopic xenograft model were produced in a previous study [ 13 ].…”

“…The BXD-1425EPN and DKFZ-EP1NS were previously derived and characterised by Dr Xiao-Nan Li, Baylor College of Medicine [ 38 ] and Dr Till Milde, DKFZ Heidelberg [ 39 ] respectively. All cell lines were grown as adherent monolayers in ‘tumour media’ as previously described [ 5 ]. The DKFZ-EP1 neurospheres (DKFZ-EP1NS) were cultured in ‘stem cell media’ as described [ 39 ].…”

“…All the reagents used in this study were bought from Sigma (St. Louis, MO, USA). Chemotherapy drugs were used at previously established IC 50 concentrations (etoposide 19.2 µM and vincristine 23.5 nM) [ 5 ], dual PPAR-(γ/δ) antagonist FH535, the PPAR-δ antagonist GSK0660, and the PPAR-γ antagonist GW9662 were all used at 15 µM and DHA was used at 20 µM.…”

“…Forty percent of cases remain incurable and 5-year survival in infants with ependymomas stands at a dismal 40–52% [ 2 , 3 ]. Thus far, only complete surgical resection has been regarded as a positive marker of clinical outcome in ependymomas [ 4 ], with genetic studies yielding just three other progostic markers, namely ABCB1 [ 5 ], gain of chromosome 1 q [ 6 , 7 ] and the RELA fusion, which specifically occurs in aggressive supratentorial ependymomas [ 8 , 9 ].…”